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KiNativ™ Application Note WWW.ACTIVXPROBES.COM KiNativ™ Application Note WWW.ACTIVXPROBES.COM ATPase Profiling . ATPase Profiling Active site profiling of ATPases and related nucleotide binding proteins Active site profiling of ATPases and related nucleotide binding proteins
The human genome contains hundreds of ATP-dependent enzymes # profiled Profiling Heat Shock Protein Inhibitors that represent potential drug targets. ActivX Biosciences’ Enzyme Class members Geldanamycin 17-AAG Radicicol HSP90 4 KiNativ™ platform has been recently expanded to provide for Enzyme Name 20μM 2μM 200nM 20nM 20μM 2μM 200nM 20nM 10μM 1μM 100nM 10nM The abundance of HSP70 9 the profiling of approximately 180 of these enzymes. The technology HSP90 , HSP90 98.5 96.3 45 -0.2 94.2 56.2 3.3 -5.3 98.8 98.7 28.1 1.6 HSP90 isoforms in a HSP60 (Chaperonin) 9 HSP90 , HSP90 96.9 95.9 50.4 5.1 95.5 73.4 10.2 -8.7 98.6 98.1 27.4 -10.1 typical cell lysate is identical to the well-validated methodology used to profile protein AAA+ (ABC transporters) 17 Endoplasmin (TRA1, GRP94) 93.4 60.6 4.9 8.7 71.7 14.1 -7 -27.8 99.3 98.2 14.2 -2.4 kinases. An ATP acyl-phosphate probe is used to label the active AAA+ (Proteasomal ATPase) 6 preparation is > 100 TRAP1 (HSP75) 73.6 14.6 -5.8 -1 10.4 -1.4 -10.1 -10.9 95.3 90.7 -1.7 -0.1 sites of enzymes that bind ATP (or related nucleotides, NAD, FAD, AAA+ (Other) 29 nM, thus any inhibitor Ubiquitin-activating E1 6 BIIB021 NVP-AUY922 10μM 1μM 10μM 1μM with an affinity of at least 100 nM will SAM, etc.) directly in native cell lysates or tissues. Because labeling ATP-Grasp domain 9 Enzyme Name 100nM 10nM 100nM 10nM occurs within the active site, the relative amount of labeling is ATP-dependent helicase 14 HSP90 , HSP90 98.8 98.5 14.5 -16.3 99.2 98.8 37.4 9 titrate the total enzyme in a typical sensitive to the binding of competitive inhibitors. The relative potency Small molecule kinase 17 HSP90 , HSP90 98.4 97.8 31.1 -7.1 98.7 97.6 29.3 6 profiling experiment. Shown values were Endoplasmin (TRA1, GRP94) 91.1 24.3 -24.2 -16.1 98.4 86.7 -8.6 -23.3 and target specificity of ATP-competitive compounds against hundreds Nudix hydrolase 4 obtained in non-reducing conditions. Aminoacyl-tRNA synthetase 8 TRAP1 (HSP75) 94 64.6 -5.8 -0.1 95.3 85.7 0 -7.9 of ATPases can be assessed in a single experiment. This initial Actin 6 VER-155008 offering is being continually refined in order to cover a larger number Miscellaneous 40 The profiling of HSP90 inhibitors revealed no Enzyme Name 100uM 10uM 1uM 0.1uM IC50 HSP70-1a 94.2 86.7 58.6 19 0.66 of ATPases. Please contact us to inquire about your enzyme of interest. inhibition outside of the 4 HSP90 family members. GRP78 89.4 77.3 44.3 19 1.4 However, profiling of the HSP70 inhibitor, HSC70 95.4 84 53.8 16.1 0.86 HSP70-13 (STCH) 63.1 19.1 17.6 0.6 57.2 Labeling site validation VER-155008, showed off-target inhibition of 5 GRP75 57.3 12.2 4 -13.8 64.5 nucleotide-binding proteins in addition to inhibition Deoxycytidine kinase 94.7 70.1 9.4 -19.5 4.2 HSP90α, HSP90β p97 ATPase Adenosylhomocysteinase 68.1 18.7 4.3 -7.8 41.7 of 5 HSP70 isoforms. All compounds were profiled IPP isomerase 1, IPP isomerase 2 48.8 15.5 17.1 3.7 139 8-oxo-dGTPase 43.2 8.3 -13.9 0.6 120 3000 control against ~ 160 ATPases found in HL60 cell lysates. control Farnesyl pyrophosphate synthetase 50.6 -2.2 -7.1 -16.6 87.9 1 mM 150000 1 mM 100 μM 100 μM 10 μM 10 μM 1 μM 1 μM 2000 Profiling Biologically-Active Flavonoids 100000 Signal Signal 2 2 Epigallocatechin μ Quercetin Myricetin Legend IC50 in M MS MS Enzyme Name gallate < 5 μM 1000 α β 50000 8-oxo-dGTPase 1.2 12 77 5 - 25 μM Ap4A hydrolase 4.6 43 36 25 - 75 μM IPP isomerase 1, IPP isomerase 2 2.6 30 > 150 75 - 150 μM μ 0 0 CoenzymeA diphosphatase 1.6 53 > 150 > 150 M 86 88 90 92 94 52 54 56 58 60 Mevalonate kinase 4.9 35 23 Time (min) Time (min) Deoxycytidine kinase 34 117 97 Quercetin and myricetin are NDP kinase 3 22 105 > 150 HSP70-1a Cytidylate kinase 97 98 83 naturally occurring flavonoids Thymidine kinase 2 120 60 47 ATP concentration ATP IC50 found in many plants. They are 6000 Enzyme Name 1 mM 100 μM 10 μM 1 μM (μM) Farnesyl pyrophosphate synthetase 1.7 13.7 28 control 2',5'-phosphodiesterase 12 2.0 4.2 > 150 thought to have antioxidant, anti- 1 mM HSP90 , HSP90 96.3 91.8 24.2 -5.0 19.8 100 μM Amidophosphoribosyltransferase 8.6 > 150 > 150 inflammatory and anti-cancer 10 μM HSP70-1a 97.4 94.3 58.1 7.2 7.6 SAICAR synthetase 1.8 50 > 150 1 μM 4000 p97 ATPase (ATP site1) 99.2 98.3 68.5 33.9 2.8 tRNA-dihydrouridine synthase 87 3.7 6.3 properties through a variety of values in % inhibition DNA-directed RNA Pol III 38 126 > 150 Signal mechanisms, including ATP- 2 dCTP pyrophosphatase 8.0 13 28 MS Labeling sites for the ATP probe were validated to be active HPRTase > 150 70 33 competitive inhibition of lipid and 2000 AMPK 1 (site 2) 91 100 42 protein kinases. EGCG is a related site peptides by performing competition experiments with AMPK 1, AMPK 2 (site 1) > 150 > 150 > 150 free ATP. Using this criteria, over 240 labeling sites AMPK 2 (site 2) 72 > 150 61 flavonoid and the major bio-active Autophagy-related protein 7 > 150 77 37 0 ingredient of green tea. The 42 44 46 48 from approximately 180 proteins have been validated. Ubiquitin-activating enzyme E1 > 150 > 150 100 Time (min) NEDD8-activating enzyme E1 > 150 62 > 150 ATPase profiling results ATP-binding cassette sub-family D3 > 150 59 87 demonstrate that they have affinity AAA domain-containing protein 1 34 > 150 51 KIAA0564 25 8.1 6.2 for a variety of enzyme classes.
For more information about KiNativ™ call 858-526-2515 or visit www.activxprobes.com For more information about KiNativ™ call 858-526-2515 or visit www.activxprobes.com
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KiNativ™ Application Note WWW.ACTIVXPROBES.COM KiNativ™ Application Note WWW.ACTIVXPROBES.COM ATPase Profiling . ATPase Profiling Active site profiling of ATPases and related nucleotide binding proteins Active site profiling of ATPases and related nucleotide binding proteins
The human genome contains hundreds of ATP-dependent enzymes # profiled Profiling Heat Shock Protein Inhibitors that represent potential drug targets. ActivX Biosciences’ Enzyme Class members Geldanamycin 17-AAG Radicicol HSP90 4 KiNativ™ platform has been recently expanded to provide for Enzyme Name 20μM 2μM 200nM 20nM 20μM 2μM 200nM 20nM 10μM 1μM 100nM 10nM The abundance of HSP70 9 the profiling of approximately 180 of these enzymes. The technology HSP90 , HSP90 98.5 96.3 45 -0.2 94.2 56.2 3.3 -5.3 98.8 98.7 28.1 1.6 HSP90 isoforms in a HSP60 (Chaperonin) 9 HSP90 , HSP90 96.9 95.9 50.4 5.1 95.5 73.4 10.2 -8.7 98.6 98.1 27.4 -10.1 typical cell lysate is identical to the well-validated methodology used to profile protein AAA+ (ABC transporters) 17 Endoplasmin (TRA1, GRP94) 93.4 60.6 4.9 8.7 71.7 14.1 -7 -27.8 99.3 98.2 14.2 -2.4 kinases. An ATP acyl-phosphate probe is used to label the active AAA+ (Proteasomal ATPase) 6 preparation is > 100 TRAP1 (HSP75) 73.6 14.6 -5.8 -1 10.4 -1.4 -10.1 -10.9 95.3 90.7 -1.7 -0.1 sites of enzymes that bind ATP (or related nucleotides, NAD, FAD, AAA+ (Other) 29 nM, thus any inhibitor Ubiquitin-activating E1 6 BIIB021 NVP-AUY922 10μM 1μM 10μM 1μM with an affinity of at least 100 nM will SAM, etc.) directly in native cell lysates or tissues. Because labeling ATP-Grasp domain 9 Enzyme Name 100nM 10nM 100nM 10nM occurs within the active site, the relative amount of labeling is ATP-dependent helicase 14 HSP90 , HSP90 98.8 98.5 14.5 -16.3 99.2 98.8 37.4 9 titrate the total enzyme in a typical sensitive to the binding of competitive inhibitors. The relative potency Small molecule kinase 17 HSP90 , HSP90 98.4 97.8 31.1 -7.1 98.7 97.6 29.3 6 profiling experiment. Shown values were Endoplasmin (TRA1, GRP94) 91.1 24.3 -24.2 -16.1 98.4 86.7 -8.6 -23.3 and target specificity of ATP-competitive compounds against hundreds Nudix hydrolase 4 obtained in non-reducing conditions. Aminoacyl-tRNA synthetase 8 TRAP1 (HSP75) 94 64.6 -5.8 -0.1 95.3 85.7 0 -7.9 of ATPases can be assessed in a single experiment. This initial Actin 6 VER-155008 offering is being continually refined in order to cover a larger number Miscellaneous 40 The profiling of HSP90 inhibitors revealed no Enzyme Name 100uM 10uM 1uM 0.1uM IC50 HSP70-1a 94.2 86.7 58.6 19 0.66 of ATPases. Please contact us to inquire about your enzyme of interest. inhibition outside of the 4 HSP90 family members. GRP78 89.4 77.3 44.3 19 1.4 However, profiling of the HSP70 inhibitor, HSC70 95.4 84 53.8 16.1 0.86 HSP70-13 (STCH) 63.1 19.1 17.6 0.6 57.2 Labeling site validation VER-155008, showed off-target inhibition of 5 GRP75 57.3 12.2 4 -13.8 64.5 nucleotide-binding proteins in addition to inhibition Deoxycytidine kinase 94.7 70.1 9.4 -19.5 4.2 HSP90α, HSP90β p97 ATPase Adenosylhomocysteinase 68.1 18.7 4.3 -7.8 41.7 of 5 HSP70 isoforms. All compounds were profiled IPP isomerase 1, IPP isomerase 2 48.8 15.5 17.1 3.7 139 8-oxo-dGTPase 43.2 8.3 -13.9 0.6 120 3000 control against ~ 160 ATPases found in HL60 cell lysates. control Farnesyl pyrophosphate synthetase 50.6 -2.2 -7.1 -16.6 87.9 1 mM 150000 1 mM 100 μM 100 μM 10 μM 10 μM 1 μM 1 μM 2000 Profiling Biologically-Active Flavonoids 100000 Signal Signal 2 2 Epigallocatechin μ Quercetin Myricetin Legend IC50 in M MS MS Enzyme Name gallate < 5 μM 1000 α β 50000 8-oxo-dGTPase 1.2 12 77 5 - 25 μM Ap4A hydrolase 4.6 43 36 25 - 75 μM IPP isomerase 1, IPP isomerase 2 2.6 30 > 150 75 - 150 μM μ 0 0 CoenzymeA diphosphatase 1.6 53 > 150 > 150 M 86 88 90 92 94 52 54 56 58 60 Mevalonate kinase 4.9 35 23 Time (min) Time (min) Deoxycytidine kinase 34 117 97 Quercetin and myricetin are NDP kinase 3 22 105 > 150 HSP70-1a Cytidylate kinase 97 98 83 naturally occurring flavonoids Thymidine kinase 2 120 60 47 ATP concentration ATP IC50 found in many plants. They are 6000 Enzyme Name 1 mM 100 μM 10 μM 1 μM (μM) Farnesyl pyrophosphate synthetase 1.7 13.7 28 control 2',5'-phosphodiesterase 12 2.0 4.2 > 150 thought to have antioxidant, anti- 1 mM HSP90 , HSP90 96.3 91.8 24.2 -5.0 19.8 100 μM Amidophosphoribosyltransferase 8.6 > 150 > 150 inflammatory and anti-cancer 10 μM HSP70-1a 97.4 94.3 58.1 7.2 7.6 SAICAR synthetase 1.8 50 > 150 1 μM 4000 p97 ATPase (ATP site1) 99.2 98.3 68.5 33.9 2.8 tRNA-dihydrouridine synthase 87 3.7 6.3 properties through a variety of values in % inhibition DNA-directed RNA Pol III 38 126 > 150 Signal mechanisms, including ATP- 2 dCTP pyrophosphatase 8.0 13 28 MS Labeling sites for the ATP probe were validated to be active HPRTase > 150 70 33 competitive inhibition of lipid and 2000 AMPK 1 (site 2) 91 100 42 protein kinases. EGCG is a related site peptides by performing competition experiments with AMPK 1, AMPK 2 (site 1) > 150 > 150 > 150 free ATP. Using this criteria, over 240 labeling sites AMPK 2 (site 2) 72 > 150 61 flavonoid and the major bio-active Autophagy-related protein 7 > 150 77 37 0 ingredient of green tea. The 42 44 46 48 from approximately 180 proteins have been validated. Ubiquitin-activating enzyme E1 > 150 > 150 100 Time (min) NEDD8-activating enzyme E1 > 150 62 > 150 ATPase profiling results ATP-binding cassette sub-family D3 > 150 59 87 demonstrate that they have affinity AAA domain-containing protein 1 34 > 150 51 KIAA0564 25 8.1 6.2 for a variety of enzyme classes.
For more information about KiNativ™ call 858-526-2515 or visit www.activxprobes.com For more information about KiNativ™ call 858-526-2515 or visit www.activxprobes.com
® ®